The present invention is in support of the development of an oleaginous yeast that accumulates oils enriched in long-chain ω-3 and/or ω-6 polyunsaturated fatty acids (“PUFAs”; e.g., 18:3, 18:4, 20:3, 20:4, 20:5, 22:6 fatty acids). Toward this end, the natural abilities of oleaginous yeast (mostly limited to 18:2 fatty acid production) have been enhanced by advances in genetic engineering, leading to the production of 20:4 (arachidonic acid or “ARA”), 20:5 (eicosapentaenoic acid or “EPA”) and 22:6 (docosahexaenoic acid or “DHA”) PUFAs in transformant Yarrowia lipolytica. These ω-3 and ω-6 fatty acids were produced by introducing and expressing heterologous genes encoding the ω-3/ω-6 biosynthetic pathway in the oleaginous host (see W2004/101757 and co-pending U.S. Patent Application No. 60/624,812). However, in addition to developing techniques to introduce the appropriate fatty acid desaturases and elongases into these particular host organisms, it is also necessary to increase the transfer of PUFAs into storage lipid pools following their synthesis.
As is well known in the art, the process of triacylglycerol (TAG) biosynthesis (wherein newly synthesized PUFAs are transferred into a host organism's storage lipid pools) requires the catalytic activity of various acyltransferases as most free fatty acids become esterified to coenzyme A (CoA) to yield acyl-CoAs. Specifically, a series of four reactions occur in the endoplasmic reticulum of the cell to form TAGs, as shown in the Table below.
TABLE 1General Reactions Of de Novo Triacylglycerol BiosynthesisReactionEnzymesn-Glycerol-3-PhosphateGlycerol-3-phosphate acyltransferase (GPAT);→ Lysophosphatidic[E.C. 2.3.1.15]; esterifies 1st acyl-CoA to sn-1Acid (1-acyl-sn-position of sn-glycerol 3-phosphateglycerol 3-phosphate or“LPA”)LPA → PhosphatidicLysophosphatidic acid acyltransferaseAcid (1,2-diacylglycerol(LPAAT) [E.C. 2.3.1.51]; esterifies 2ndphosphate or “PA”)acyl-CoA to sn-2 position of LPAPA → 1,2-Phosphatidic acid phosphatase [E.C. 3.1.3.4]Diacylglycerol (“DAG”)removes a phosphate from PADAG → TriacylglycerolDiacylglycerol acyltransferase (DGAT) [E.C.(“TAG”)2.3.1.20]; transfers acyl-CoA to the sn-3position of DAGOrPhospholipid:diacylglycerol acyltransferase(PDAT) [E.C.2.3.1.158]; transfers fattyacyl-group from sn-2 position ofphosphatidylcholine to sn-3 position ofDAGIn addition to those acyltransferases above, acyl-CoA:cholesterol acyltransferases (ACATs), lecithin:cholesterol acyltransferases (LCATs) and lysophosphatidylcholine acyltransferases (LPCATs) are also intimately involved in the biosynthesis of TAGs. The role of each of these acyltransferases in regulating lipid acyl composition is largely mediated through their individual substrate specificities.
This application is concerned primarily with the first step in the synthesis of TAG (wherein glycerol-3-phosphate is converted to LPA), thereby limiting the acyltransferase(s) of primary importance to GPAT (also referred to as glycerol-3-phosphate o-acyltransferase in the literature). GPAT activity is found in all species including bacteria, fungi, plants and animals. In mammals, it is found to varying degrees in many tissues including liver, adipose, heart, lung, kidney, adrenal, muscle, lactating mammary, intestinal mucosa, brain and in many mammalian cultured cell lines (Bell, R. M., et al., In: The Enzymes, (Boyer, P. D., ed.) v. 16, pp. 87–112, Academic NY (1983)). There are two known isoforms of GPAT activity in mammals: one which isolates with the mitochondria, preferentially uses saturated fatty acyl-CoAs and whose major acylation end product is primarily LPA; and, one which isolates with the microsomal endoplasmic reticulum (ER) fraction, uses saturated and unsaturated fatty acyl-CoAs equally well and whose major acylation product is PA (Hill, J. O., et al. Science 280:1371–1374 (1998); Dircks, L., Sul, H. S., Lipid Res. 38:461–479 (1999)). Similarly, the plant cell contains three types of GPAT, which are located in the chloroplasts, mitochondria and cytoplasm, respectively. The enzyme in chloroplasts is soluble and uses acyl-(acyl-carrier protein) as the acyl donor, whereas the enzymes in the mitochondria and the cytoplasm are bound to membranes and use acyl-CoA as the acyl donor. The distinct fatty-acyl preferences of these various GPAT isoforms is thought to be responsible for the observed predominance of saturated (versus unsaturated) fatty acids in the sn-1 position. GPAT is also potentially a rate-limiting reaction, and thus should be considered an important and controlling enzyme early in the pathway of de novo synthesis of TAGs and phospholipids.
Despite the clear importance of GPAT in glycerophospholipid biosynthesis, characterization of different GPAT isoforms has been difficult and sequence information (either nucleotide or protein) of GPAT genes is limited. It is predicted that a GPAT from a microorganism that naturally produces long-chain PUFAs (e.g., Mortierella, Pythium, Cyclotella, Nitzschia, Crypthecodinium and Thraustochytrium, producing e.g., ARA, EPA and/or DHA) would incorporate long-chain PUFAs with increased efficiency, relative to a GPAT that does not naturally interact with long-chain PUFAs. The only known disclosure providing genes encoding GPATs from these types of organisms is that of WO 2004/087902 (describing GPATs in the moss, Physcomitrella patens). The microsomal GPAT of Mortierella ramanniana var. angulispora was recently purified to homogeneity and its acyl-CoA specificity was characterized (Mishra, S., Biochem. J. 355(10):315–322 (2001))]; however, the protein was not sequenced. Thus, there is a need for the identification and isolation of a gene encoding GPAT from an organism such as those suggested above, to permit its use in the production and accumulation of long-chain PUFAs in the storage lipid pools (i.e., TAG fraction) of transformant oleaginous yeast.
Surprisingly, the Applicants have isolated a novel GPAT gene from the filamentous fungus Mortierella alpina. It is expected that the gene of the present invention (“GPAT”) will be useful to enable one to modify the transfer of long-chain free fatty acids (e.g., ω-3 and/or ω-6 fatty acids) into the TAG pool in oleaginous yeast.